The polyamines putrescine, spermidine and spermine are thought to have significant regulatory roles in cell metabolism. We intend to study the synthesis and function of polyamines in Trypanosoma brucei and attempt to determine their critical functions in the parasite. From studies in the literature and our work, we believe polyamines may be of special importance to trypanosomatids and may be a specific target of existing cationic trypanocides. This concept may be especially significant since we cured T. brucei mouse infections with alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (a key polyamine synthetic enzyme). The proposal aims at clarifying the synthesis, function, and pharmacologic potential of polyamines in trypanosomes. We intend to study: a) synthetic pathways and control of synthesis (via ornithine decarboxylase) in blood and culture forms of T. brucei; b) intracellular target sites, including DNA polymerase and NAD-linked alpha-glycerophosphate dehydrogenase (we found both are polyamine-activated); c) the relationship of polyamines to cationic trypanocides, both in vitro and in vivo (we have demonstrated that coadministration of polyamine with trypanocides in vivo negates drug action). These studies will combine in vivo, in vitro (whole cell), subcellular and purified enzyme analyses, and will make extensive use of radiotracer techniques and thin layer chromatography. Ion exchange and affinity chromatography would be used for purification of T. brucei DNA polymerase. We would continue to investigate polyamine-trypanocide interactions in vivo, by combination therapy using low doses of trypanocides plus polyamine antagonists. We hope, by these studies, to clarify the activity of polyamines on trypanosomes, to uncover additional target sites for chemotherapeutic attack, and to develop novel agents or combinations of agents potentially useful in clinical and veterinary trypanosomiasis.